This proposal outlines experiments that will provide mechanistic insights into and accelerate the medicinal development of small molecules that perturb the function of the ClpP peptidase in M. tuberculosis, the deadliest bacterial pathogen. The Mtb ClpP peptidase is an atypically complex, barrel-shaped assemblage of fourteen subunits that degrades proteins through associations with ATP-dependent chaperones that recognize and unfold substrates. In the past five years, my collaborator, Prof. Robert Sauer at MIT and I have made significant investments of time and effort in the functional reconstitution of the heterotetradecameric ClpP and accessory ATPases (ClpX and ClpC1) from Mtb. With that success, we have been able to characterize our rationally designed modulators of ClpP activity. The voluminous preliminary data (much of which is published) that we have generated provide a clear roadmap for the proposed research. They will yield insights into protein homeostasis in bacteria that can be exploited in drug development and molecules that could be much needed additions to the dwindling armamentarium used in the fight against multi-drug resistant Mtb. Aim 1. Develop and characterize small molecule modulators of the Mtb ClpP system. This aim is centered in chemical synthesis and enzymology. One objective is the multi-gram synthesis of an optimized, mycobactericidal ADEP that inhibits the ClpP system for use in murine models of tuberculosis. Another is optimization of a novel ADEP fragment that kills Mtb by activation of its ClpP system. We will also synthesize rationally designed ClpP inhibitors lacking the pharmacological liabilities of the only known mechanism-based inhibitor (developed in our laboratories). Compounds will be evaluated in in vitro assays of Clp system activity. Aim 2. Assess activities of and resistance to ClpP modulators in living cultures of mycobacteria. This aim is focused on comparative evaluations of ClpP modulators in living cultures of mycobacteria- including assays of minimal inhibitory and bactericidal concentrations. We believe that identification of the substrates of the Mtb Clp system will reveal insights into the mechanisms of these compounds. By applying new proteomics technologies along with a well-established in vivo ClpP substrate trapping method to the M. smegmatis Clp system for the first time, we will identify its substrates in the protease's native state and as it is perturbed by modulators. We will also investigate the mechanisms of small molecules that potentiate ADEP activity against Mtb by as much as 16-fold (i.e., hypothetical suppressors of efflux and the tuberculosis drug bedaquiline). Aim 3. Assess activities of ClpP modulators in mouse models of tuberculosis. Though ADEPs cured certain bacterial infections in animals, they have not been studied in tuberculosis. In murine models of tuberculosis, Dr. William Bishai at Johns Hopkins will assess the efficacies of an optimized ADEP alone and in combination with bedaquiline, standard tuberculosis drug regimens, and a potentiator that we designed to act by suppression of ADEP efflux by Mtb. Analogous experiments will be performed with our Mtb ClpP activator.